Adult periodontitis is characterized by a reduction in fiber density of gingival collagen, a major component of human gingival tissues. These collagen fibers form the periodontal ligament that attaches the tooth to the alveolar bone. Destruction of the collagen fibers, therefore, destabilizes the tooth in the bone and contributes to tooth loss. The destruction of collagen is probably due to the action of the enzyme, collagenase, which is produced by various host cells and/or bacterial cells. The anaerobic oral bacterium, Bacteroides gingivalis, which had been implicated as a major pathogen in adult periodontitis, produces collagenase, while other oral Bacteroides spp. do not. Therefore, the production of collagenase by B. gingivalis may be an important virulence factor for this organism. This study is designed to assess the role of collagenase in the pathogenicity of B gingivalis. The central aim of this study is to construct isogenic strains of B. gingivalis differing only in the ability to produce collagenase. Two approaches will be used to construct these strains: 1) chemical and/or transpositional mutagenesis in vivo of B. gingivalis, and 2) in vitro mutagenesis by deletion or insertion into a cloned B. gingivalis collagenase gene. Preliminary experiments in characterizing the B. gingivalis collagenases, e.g., determination of molecular weight, production as a proenzyme, and monomer vs. multimer structure, will be helpful in devising the best cloning strategy and identifying the lesions in in vivo generated B. gingivalis mutants. The cloned B. gingivalis gene will be used as a probe in DNA-DNA hybiridzation to determine the prevalence of this gene in oral Bacteroides spp. Although B. gingivalis is considered to be the major periodontal pathogen among the Bacteroides ssp., other black pigmented Bacteroides spp. have been implicated. If these latter strains contain the collagenase gene, while Bacteroides spp. from healthy individuals do not, then the collagenase can be considered to be an important virulence factor. The isogenic B. gingivalis strains, differing only to collagenase production, will be used in a variety of in vitro model systems. These experiments will be performed in collaboration with other investigators involved in the Research Center in Oral Biology. These experiments and future studies of the virulence of the isogenic strains in vivo should allow us to evaluate the importance of collagenase in the pathogenicity of black pigmented oral Bacteroides spp.